ABCMdb

ABCB1 p.Arg580Cys

Predicted by SNAP2:	A: D (85%), C: D (63%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (75%), I: D (85%), K: D (71%), L: D (91%), M: D (75%), N: D (91%), P: D (91%), Q: D (80%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Cysteine-scanning mutagenesis provides no evidence... EMBO J. 1999 Dec 1;18(23):6800-8. Blott EJ, Higgins CF, Linton KJ
Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.
EMBO J. 1999 Dec 1;18(23):6800-8., 1999-12-01 [PMID:10581253]

Abstract [show]
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.

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No. Sentence Comment
39 Four SC mutants (P549C, V569C, K578C and R580C) were expressed at significantly lower levels than wild-type P-gp, but use of an alternative transfection reagent (lipofectamine) allowed sufficient expression for further analysis. Login to comment
59 Cells were transiently transfected with the relevant cDNA, using lipofectin or lipofectamine (for SC mutants P549C, V569C, K578C and R580C), and 75 µg of whole cell lysate (150 µg for K578C and R580C) was added per lane. Login to comment

[hide] Communication between the nucleotide binding domai... Biochemistry. 2003 Jul 1;42(25):7780-9. Gabriel MP, Storm J, Rothnie A, Taylor AM, Linton KJ, Kerr ID, Callaghan R
Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508.
Biochemistry. 2003 Jul 1;42(25):7780-9., 2003-07-01 [PMID:12820887]

Abstract [show]
Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.

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No. Sentence Comment
148 No recombinant virus could be obtained for R580C despite several independent cotransfection experiments. Login to comment